Combination therapy for treatment of fibrotic disorders

ABSTRACT

The present invention provides methods of treating a fibrotic disorder. The methods generally involve administering an effective amount of IFN-γ and an effective amount of pirfenidone or a pirfenidone analog.

FIELD OF THE INVENTION

This invention is in the field of therapy of treating fibrotic diseases.

BACKGROUND OF THE INVENTION

Current data indicate that fibrosis is not a static process;extracellular matrix is constantly being laid down and resorbed and theprogressive accumulation of fibrous tissue is thought to represent arelative imbalance between pro-fibrotic processes and anti-fibroticprocesses. If these processes are not properly regulated, the pathologicand progressive accumulation of collagen in the extracellular space as aresult of a disordered wound healing process leads to replacement ofnormal cells by dense fibrous bands of protein, and results in fibroticdisease with disordered function in the affected organ (for example,impairment of respiratory function, impaired circulatory function viafibrotic changes in arterial walls, fibrotic degeneration of renal andliver function, degenerative musculoskeletal function, fibroticdegeneration of cardiac muscle or skeletal muscle, fibrotic degenerativechanges in neuronal tissues in the central nervous system as well as theperipheral nervous system, etc.).

Pulmonary fibrosis can be caused by a number of different conditions,including sarcoidosis, hypersensitivity pneumonitis, collagen vasculardisease, and inhalant exposure. The diagnosis of these conditions canusually be made by careful history, physical examination, chestradiography, including a high resolution computer tomographic scan(HRCT), and transbronchial biopsies. However, in a significant number ofpatients, no underlying cause for the pulmonary fibrosis can be found.These conditions of unknown etiology have been termed idiopathicinterstitial pneumonias or idiopathic pulmonary fibrosis. Histologicexamination of tissue obtained at open lung biopsy allows classificationof these patients into several categories, including Usual InterstitialPneumonia (UIP), Desquamative Interstitial Pneumonia (DIP), andNon-Specific Interstitial Pneumonia (NSIP).

The logic of dividing idiopathic interstitial pneumonias into thesecategories is based not only on histology, but also on the differentresponse to therapy and prognosis for these different entities. DIP isassociated with smoking and the prognosis is good, with more than 70% ofthese patients responding to treatment with corticosteroids. NSIPpatients are also frequently responsive to steroids and prognosis isgood, with 50% of patients surviving to 15 years. In contrast, the UIPhistologic pattern is associated with a poor response to therapy and apoor prognosis, with survival of only 3-5 years.

Idiopathic pulmonary fibrosis (IPF) is the most common form ofidiopathic interstitial pneumonia and is characterized by the UIPpattern on histology. IPF has an insidious onset, but once symptomsappear, there is a relentless deterioration of pulmonary function anddeath within 3-5 years after diagnosis. The mean age of onset is 60-65and males are affected approximately twice as often as females.Prevalence estimates are 13.2-20.2 per 100,000. The annual incidence isestimated to be 7.4-10.7 per 100,000 new cases per year.

Published evidence suggests that less than 20% of patients with IPFrespond to steroids. In patients who have failed treatment withsteroids, cytotoxic drugs such as azathioprine or cyclophosphamide aresometimes added to the steroid treatment. However, a large number ofstudies have shown little or no benefit of these drugs. There arecurrently no drugs approved for treatment of IPF.

Fibrosis of the liver occurs due to a chronic toxic insult to the liversuch as hepatitis C virus (HCV) or hepatitis B virus (HBV) infection,autoimmune injury, and chronic exposure to toxins such as alcohol.Chronic toxic insult leads to repeated cycles of hepatocyte injury andrepair accompanied by chronic inflammation. Over a variable period oftime, abnormal extracellular matrix progressively accumulates as aconsequence of the host's wound repair response. Left unchecked, thisleads to increasing deposition of fibrous material until liverarchitecture becomes distorted and the liver's regenerative ability iscompromised. The progressive accumulation of scar tissue within theliver finally results in the histopathologic picture of cirrhosis,defined as the formation of fibrous septae throughout the liver with theformation of micronodules.

Renal fibrosis is a complication of kidney injury and can contribute toorgan failure. Tubulointerstitial and glomerular fibrosis is amorphologic hallmark of chronic, progressive renal disease and isthought to be the final common mechanism leading to end-stage renaldisease. There are multiple etiologies of renal fibrosis. In particular,Type I and II diabetes mellitus are common causes of renal fibrosis. Inaddition, there are toxic, drug-induced, metabolic, structural, genetic,and infectious causes of chronic renal insufficiency related to renalfibrosis. In a number of pathologic conditions, the etiology is unknown.Of particular clinical relevance, the rate of decline of the glomerularfiltration rate in patients with chronic renal disease correlatesstrongly with the extent of tubulointerstitial and glomerular injury.Tubulointerstitial fibrosis is also a component of age-relatedstructural changes in otherwise normal kidneys and is a hallmark ofchronic allograft nephropathy (chronic allograft rejection), the mostcommon cause of kidney transplant failure in the first decade aftertransplantation. Accumulation of proteins, such as fibronectin andvarious collagens, in the interstitium of the kidney is thought to be afundamental process in development of tubulointerstitial scarring.Increased synthesis, decreased degradation, or both can underlieinterstitial protein accumulation. During fibrosis, interstitialfibroblasts proliferate and are primarily responsible for increasedproduction of interstitial proteins. Currently, there are no drugs thatadequately treat renal fibrosis.

In addition to fibrotic disorders of the lung, liver and kidney, manyother organs and tissues are susceptible to fibrotic degeneration. Inparticular, cardiac injury from hypoxia or ischemia, toxins, infectiousagents, genetic etiologies, and structural disorders can lead to aninappropriate chronic wound healing process that results in fibrosis ofcardiac tissue.

There is a need in the art for methods of treating fibrotic disorders.The present invention addresses this need.

LITERATURE

WO 01/34180; Ziesche et al. (1999) N. Engl. J. Med. 341:1264-1269; duBois (1999) N. Engl. J. Med. 341:1302-1304; U.S. Pat. No. 6,294,350; EP795,332; King (2000) N. Engl. J. Med. 342:974-975; Ziesche and Block(2000) Wien. Klin Wochenschr. 112:785-790; Raghu et al. (1999) Am. J.Respir. Crit. Care Med. 159:1061-1069; Stem et al. (2001) Chest120:213-219; Gay et al. (1998) Am. J. Respir. Crit. Care Med.157:1063-1072; Dayton et al. (1993) Chest 103:69-73.

Al-Bayati et al. (2002) Biochem. Pharmacol. 64:517-525; Shihab et al.(2002) Am. J. Transplant. 2:111-119; Yu et al. (2002) Curr. OpinionPharmacol. 2:177-181; U.S. Pat. Nos. 5,310,562; 5,518,729; 5,716,632;and 6,090,822.

METAVIR (1994) Hepatology 20:15-20; Brunt (2000) Hepatol. 31:241-246;Alpini (1997) J. Hepatol. 27:371-380; Baroni et al. (1996) Hepatol. 23:1189-1199; Czaja et al. (1989) Hepatol. 10:795-800; Grossman et al.(1998) J. Gastroenterol. Hepatol. 13:1058-1060; Rockey and Chung (1994)J. Invest. Med. 42:660-670; Sakaida et al. (1998) J. Hepatol. 28 :471479; Shi et al. (1997) Proc. Natl. Acad. Sci. USA 94:10663-10668; Baroni etal. (1999) Liver 19:212-219; Lortat-Jacob et al. (1997) J. Hepatol.26:894-903; Llorent et al. (1996) J. Hepatol. 24:555-563.

SUMMARY OF THE INVENTION

The present invention provides methods of treating fibrotic diseaseswith a combination therapy of IFN-γ and pirfenidone or specificpirfenidone analogs. The methods generally involve administering to anindividual suffering from a fibrotic disorder a therapeuticallyeffective amount of IFN-γ in combination with a therapeuticallyeffective amount of pirfenidone or a specific pirfenidone analog. Inparticular, the methods of the invention involve administering to anindividual suffering from a fibrotic disorder a synergistic combinationof IFN-i and pirfenidone or a specific pirfenidone analog.

DEFINITIONS

As used herein, the terms “treatment”, “treating”, and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) increasing-survival time; (b) decreasing the risk of deathdue to the disease; (c) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (d) inhibiting the disease, i.e., arresting itsdevelopment (e.g., reducing the rate of disease progression); and (e)relieving the disease, i.e., causing regression of the disease.

The terms “individual,” “host,” “subject,” and “patient,” usedinterchangeably herein, refer to a mammal, particularly a human.

The term “therapeutically effective amount” is meant an amount of atherapeutic agent, or a rate of delivery of a therapeutic agent,effective to facilitate a desired therapeutic effect. The precisedesired therapeutic effect will vary according to the condition to betreated, the formulation to be administered, and a variety of otherfactors that are appreciated by those of ordinary skill in the art.

A “fibrotic condition,” “fibrotic disease” and “fibrotic disorder” areused interchangeably to refer to a condition, disease or disorder thatis amenable to treatment by administration of a compound havinganti-fibrotic activity. Fibrotic disorders include, but are not limitedto, pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF)and pulmonary fibrosis from a known etiology, liver fibrosis, andrenal-fibrosis. Other exemplary fibrotic conditions includemusculoskeletal fibrosis, cardiac fibrosis, post-surgical adhesions,scleroderma, glaucoma, and skin lesions such as keloids.

A “specific pirfenidone analog,” and all grammatical variants thereof,refers to, and is limited to, each and every pirfenidone analog shown inTable 1.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise.

Thus, for example, reference to “a method” includes a plurality of suchmethods and reference to “an IFN-γ dose” includes reference to one ormore doses and equivalents thereof known to those skilled in the art,and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating fibrotic diseases,including pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF),pulmonary fibrosis from a known etiology, liver fibrosis, cardiacfibrosis, and renal fibrosis. The methods generally involveadministering a therapeutically effective combination of IFN-γ andpirfenidone or a specific pirfenidone analog to an individual with afibrotic disease. In particular, the methods of the invention involveadministering to an individual suffering from fibrotic disease asynergistic combination of IFN-γ and pirfenidone or a specificpirfenidone analog.

Methods of Treating Fibrotic Diseases

The present invention provides methods for treating a fibrotic disorderin an individual having a fibrotic disorder. The method generallyinvolves administering an effective amount of interferon-gamma (IFN-γ),and an effective amount of pirfenidone or a specific analog thereof. Themethods provide for treatment of fibrotic diseases, including thoseaffecting the lung such as idiopathic pulmonary fibrosis, pulmonaryfibrosis from a known etiology, liver fibrosis or cirrhosis, cardiac andrenal fibrosis. The etiology may be due to any acute or chronic insultincluding toxic, metabolic, genetic and infectious agents.

Fibrosis is generally characterized by the pathologic or excessiveaccumulation of collagenous connective tissue. Fibrotic disordersinclude, but are not limited to, collagen disease, interstitial lungdisease, human fibrotic lung disease (e.g., obliterative bronchiolitis,idiopathic pulmonary fibrosis, pulmonary fibrosis from a known etiology,tumor stroma in lung disease, systemic sclerosis affecting the lungs,Hermansky-Pudlak syndrome, coal worker's pneumoconiosis, asbestosis,silicosis, chronic pulmonary hypertension, AIDS-associated pulmonaryhypertension, sarcoidosis, and the like), fibrotic vascular disease,arterial sclerosis, atherosclerosis, varicose veins, coronary infarcts,cerebral infarcts, myocardial fibrosis, musculoskeletal fibrosis,post-surgical adhesions, human kidney disease (e.g., nephritic syndrome,Alport's syndrome, HIV-associated nephropathy, polycystic kidneydisease, Fabry's disease, diabetic nephropathy, chronicglomerulonephritis, nephritis associated with systemic lupus, and thelike), cutis keloid formation, progressive systemic sclerosis (PSS),primary sclerosing cholangitis (PSC), liver fibrosis, liver cirrhosis,renal fibrosis, pulmonary fibrosis, cystic fibrosis, chronic graftversus host disease, scleroderma (local and systemic), Grave'sopthalmopathy, diabetic retinopathy, glaucoma, Peyronie's disease, penisfibrosis, urethrostenosis after the test using a cystoscope, inneraccretion after surgery, scarring, myelofibrosis, idiopathicretroperitoneal fibrosis, peritoneal fibrosis from a known etiology,drug-induced ergotism, fibrosis incident to benign or malignant cancer,fibrosis incident to microbial infection (e.g., viral, bacterial,parasitic, fungal, etc.), Alzheimer's disease, fibrosis incident toinflammatory bowel disease (including stricture formation in Crohn'sdisease and microscopic colitis), fibrosis induced by chemical orenvironmental insult (e.g., cancer chemotherapy, pesticides, radiation(e.g., cancer radiotherapy), and the like), and the like.

In some embodiments, an effective amount of IFN-γ and an effectiveamount of pirfenidone or a specific pirfenidone analog are amounts that,when administered in combination therapy, are effective to reducefibrosis by at least about 10%, at least about 15%, at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, or at least about 50%, or more, comparedwith the degree of fibrosis in the individual prior to treatment withthe combination therapy.

In some embodiments, an effective amount of IFN-γ and an effectiveamount of pirfenidone or a specific pirfenidone analog are amounts that,when administered in combination therapy, are effective to increase atleast one function of the organ affected by fibrosis (e.g., lung, liver,kidney, etc.) by at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, or at least about 50%, or more,compared to the basal level of organ function in the individual prior totreatment with the combination therapy.

Methods of measuring the extent of fibrosis in a given organ, andmethods of measuring the function of any given organ, are well known inthe art.

In some embodiments, the present invention provides methods of treatinga fibrotic condition that involve administering a synergisticcombination of IFN-γ and pirfenidone or specific pirfenidone analog. Asused herein, a “synergistic combination” of IFN-γ and pirfenidone or aspecific pirfenidone analog is a combined dosage that is more effectivein the therapeutic or prophylactic treatment of a fibrotic conditionthan the incremental improvement in treatment outcome that could bepredicted or expected from a merely additive combination of (i) thetherapeutic or prophylactic benefit of IFN-γ when administered at thatsame dosage as a monotherapy and (ii) the therapeutic or prophylacticbenefit of pirfenidone or a specific pirfenidone analog whenadministered at the same dosage as a monotherapy.

The invention also provides a method for treatment of a fibroticdisease, such as IPF, liver fibrosis or renal fibrosis, in an individualcomprising administering to the individual a combination of IFN-γ andpirfenidone or a specific pirfenidone analog that is effective forprophylaxis or therapy of fibrotic disease in the individual, e.g.,increasing the probability of survival, reducing the risk of death,ameliorating the disease burden or slowing the progression of disease inthe individual, while reducing the incidence or severity of one or moreside effects that would ordinarily arise from treatment with aneffective amount of IFN-γ or pirfenidone or a specific pirfenidoneanalog alone.

Methods of Treating Idiopathic Pulmonary Fibrosis

The present invention provides methods of treating idiopathic pulmonaryfibrosis (IPF). The methods generally involve administering to anindividual having IPF a combination of an effective amount of IFN-γ andan effective amount of pirfenidone or a specific pirfenidone analog.

In some embodiments, a diagnosis of IPF is confirmed by the finding ofusual interstitial pneumonia (UIP) on histopathological evaluation oflung tissue obtained by surgical biopsy. The criteria for a diagnosis ofIPF are known. Ryu et al. (1998) Mayo Clin.

Proc. 73:1085-1101.

In other embodiments, a diagnosis of IPF is a definite or probable IPFmade by high resolution computer tomography (HRCT). In a diagnosis byHRCT, the presence of the following characteristics is noted: (1)presence of reticular abnormality and/or traction bronchiectasis withbasal and peripheral predominance; (2) presence of honeycombing withbasal and peripheral predominance; and (3) absence of atypical featuressuch as micronodules, peribronchovascular nodules, consolidation,isolated (non-honeycomb) cysts, ground glass attenuation (or, ifpresent, is less extensive than reticular opacity), and mediastinaladenopathy (or, if present, is not extensive enough to be visible onchest x-ray). A diagnosis of definite IPF is made if characteristics(1), (2), and (3) are met. A diagnosis of probable IPF is made ifcharacteristics (1) and (3) are met.

In some embodiments, an “effective amount” of IFN-γ in combination withan “effective amount” of pirfenidone or a specific pirfenidone analog isa dosage combination that is effective to decrease disease progressionby at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, or more, comparedwith a placebo control or an untreated control.

In other embodiments, the present invention provides methods thatinvolve administering a synergistic combination of IFN-γ and pirfenidoneor specific pirfenidone analog. As used herein, a “synergisticcombination” of IFN-γ and pirfenidone or specific pirfenidone analog isa combined dosage that is more effective in the therapeutic orprophylactic treatment of IPF than the incremental improvement intreatment outcome that could be predicted or expected from a merelyadditive combination of (i) the therapeutic or prophylactic benefit ofIFN-γ when administered at that same dosage as a monotherapy and (ii)the therapeutic or prophylactic benefit of pirfenidone or a specificpirfenidone analog when administered at the same dosage as amonotherapy.

The invention also provides a method for treatment of IPF in anindividual comprising administering to the individual a combination ofIFN-γ and pirfenidone or a specific pirfenidone analog that is effectivefor prophylaxis or therapy of IPF in the individual, e.g., increasingthe probability of survival, reducing the risk of death, amelioratingthe disease burden or slowing the progression of disease in theindividual, while reducing the incidence or severity of one or more sideeffects that would ordinarily arise from treatment with an effectiveamount of IFN-γ or pirfenidone or a specific pirfenidone analog alone.

Disease progression is the occurrence of one or more of the following:(1) a decrease in predicted FVC of 10% or more; (2) an increase in A-agradient of 5 mm Hg or more; (3) a decrease of 15% of more in singlebreath DL_(co). Whether disease progression has occurred is determinedby measuring one or more of these parameters on two consecutiveoccasions 4 to 14 weeks apart, and comparing the value to baseline.

Thus, e.g., where an untreated or placebo-treated individual exhibits a50% decrease in FVC over a period of time, an individual administeredwith an effective combination of IFN-γ and pirfenidone or specificpirfenidone analog exhibits a decrease in FVC of 45%, about 42%, about40%, about 37%, about 35%, about 32%, about 30%, or less, over the sametime period.

In some embodiments, an “effective amount” of IFN-γ in combination withan effective amount of pirfenidone or a specific pirfenidone analog is adosage combination that is effective to increase progression-freesurvival time, e.g., the time from baseline (e.g., a time point from 1day to 28 days before beginnig of treatment) to death or diseaseprogression is increased by at least about 10%, at least about 20%, atleast about 25%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, or more, compared a placebo-treatedor an untreated control individual. Thus, e.g., in some embodiments aneffective amount of IFN-γ in combination with an effective amount ofpirfenidone or a specific pirfenidone analog is a dosage combinationthat is effective to increase the progression-free survival time by atleast about 1 week, at least about 2 weeks, at least about 3 weeks, atleast about 4 weeks, at least about 2 months, at least about 3 months,at least about 4 months, at least about 5 months, at least about 6months, at least about 8 months, at least about 10 months, at leastabout 12 months, at least about 18 months, at least about 2 years, atleast about 3 years, or longer, compared to a placebo-treated oruntreated control.

In some embodiments, an effective amount of IFN-γ in combination with aneffective amount of pirfenidone or a specific pirfenidone analog is adosage combination that is effective to increase at least one parameterof lung function, e.g., an effective amount of a combination of IFN-γand pirfenidone or a specific pirfenidone analog increases at least oneparameter of lung function by at least about 10%, at least about 20%, atleast about 25%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, or more, compared to an untreatedindividual or a placebo-treated control individual. In some of theseembodiments, a determination of whether a parameter of lung function isincreased is made by comparing the baseline value with the value at anytime point after the beginning of treatment, e.g., 48 weeks after thebeginning of treatment, or between two time points, e.g., about 4 toabout 14 weeks apart, after the beginning of treatment.

In some embodiments, an effective amount of IFN-γ in combination with aneffective amount of pirfenidone or a specific pirfenidone analog is adosage combination that is effective to increase the FVC by at leastabout 10% at least about 20%, at least about 25%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 2-fold, atleast about 3-fold, at least about 4-fold, at least about 5-fold, ormore compared to baseline on two consecutive occasions 4 to 14 weeksapart.

In some of these embodiments, an effective amount of IFN-γ incombination with an effective amount of pirfenidone or a specificpirfenidone analog is a dosage combination that results in a decrease inalveolar:arterial (A-a) gradient of at least about 5 mm Hg, at leastabout 7 mm Hg, at least about 10 mm Hg, at least about 12 mm Hg, atleast about 15 mm Hg, or more, compared to baseline.

In some of these embodiments, an effective amount of IFN-γ incombination with an effective amount of pirfenidone or a specificpirfenidone analog is a dosage combination that increases the singlebreath DL_(CO) by at least about 15%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, or more, compared to baseline. CL_(Co) is the lung diffusingcapacity for carbon monoxide, and is expressed as mL CO/mm Hg/second.

Parameters of lung function include, but are not limited to, forcedvital capacity (FVC); forced expiratory volume (FEV₁); total lungcapacity; partial pressure of arterial oxygen at rest; partial pressureof arterial oxygen at maximal exertion.

Lung function can be measured using any known method, including, but notlimited to spirometry.

Methods of Treating Liver Fibrosis

The present invention provides methods of treating liver fibrosis,including reducing clinical liver fibrosis, reducing the likelihood thatliver fibrosis will occur, and reducing a parameter associated withliver fibrosis. The methods generally involve administering acombination of an effective amount of IFN-γ and an effective amount ofpirfenidone or specific pirfenidone analog to an individual in needthereof. In particular, the invention provides methods for treatment ofliver fibrosis comprising administering a synergistic combination ofIFN-γ and pirfenidone or specific pirfenidone analog to a patient inneed thereof. Of particular interest in many embodiments is treatment ofhumans.

Liver fibrosis is a precursor to the complications associated with livercirrhosis, such as portal hypertension, progressive liver insufficiency,and hepatocellular carcinoma. A reduction in liver fibrosis thus reducesthe incidence of such complications. Accordingly, the present inventionfurther provides methods of reducing the likelihood that an individualwill develop complications associated with cirrhosis of the liver.

The present methods generally involve administering a therapeuticallyeffective combination of IFN-γ and pirfenidone or specific pirfenidoneanalog. As used herein, an “effective amount” of IFN-γ in combinationwith an “effective amount” of pirfenidone or specific pirfenidone analogis a combined dosage of IFN-γ and pirfenidone or specific pirfenidoneanalog that is effective in reducing liver fibrosis; and/or that iseffective in reducing the likelihood that an individual will developliver fibrosis; and/or that is effective in reducing a parameterassociated with liver fibrosis; and/or that is effective in reducing adisorder associated with cirrhosis of the liver.

In other embodiments, the present invention provides methods thatinvolve administering a synergistic combination of IFN-γ and pirfenidoneor specific pirfenidone analog. As used herein, a “synergisticcombination” of IFN-γ and pirfenidone or specific pirfenidone analog isa combined dosage that is more effective in the therapeutic orprophylactic treatment of liver fibrosis than the incrementalimprovement in treatment outcome that could be predicted or expectedfrom a merely additive combination of (i) the therapeutic orprophylactic benefit of IFN-γ when administered at that same dosage as amonotherapy and (ii) the therapeutic or prophylactic benefit ofpirfenidone or a specific pirfenidone analog when administered at thesame dosage as a monotherapy.

The invention also provides a method for treatment of liver fibrosis inan individual comprising administering to the individual a combinationof IFN-γ and pirfenidone or a specific pirfenidone analog that iseffective for prophylaxis or therapy of liver fibrosis in theindividual, e.g., increasing the probability of survival, reducing therisk of death, ameliorating the disease burden or slowing theprogression of disease in the individual, while reducing the incidenceor severity of one or more side effects that would ordinarily arise fromtreatment with an effective amount of IFN-γ or pirfenidone or a specificpirfenidone analog alone.

Whether treatment with a combination of IFN-γ and pirfenidone orspecific pirfenidone analog is effective in reducing liver fibrosis isdetermined by any of a number of well-established techniques formeasuring liver fibrosis and liver function. Whether liver fibrosis isreduced is determined by analyzing a liver biopsy sample. An analysis ofa liver biopsy comprises assessments of two major components:necroinflammation assessed by “grade” as a measure of the severity andongoing disease activity, and the lesions of fibrosis and parenchymal orvascular remodeling as assessed by “stage” as being reflective oflong-term disease progression. See, e.g., Brunt (2000) Hepatol.31:241-246; and METAVIR (1994) Hepatology 20:15-20. Based on analysis ofthe liver biopsy, a score is assigned. A number of standardized scoringsystems exist which provide a quantitative assessment of the degree andseverity of fibrosis. These include the METAVIR, Knodell, Scheuer,Ludwig, and Ishak scoring systems.

The METAVIR scoring system is based on an analysis of various featuresof a liver biopsy, including fibrosis (portal fibrosis, centrilobularfibrosis, and cirrhosis); necrosis (piecemeal and lobular necrosis,acidophilic retraction, and ballooning degeneration); inflammation(portal tract inflammation, portal lymphoid aggregates, and distributionof portal inflammation); bile duct changes; and the Knodell index(scores of periportal necrosis, lobular necrosis, portal inflammation,fibrosis, and overall disease activity). The definitions of each stagein the METAVIR system are as follows: score: 0, no fibrosis; score: 1,stellate enlargement of portal tract but without septa formation; score:2, enlargement of portal tract with rare septa formation; score: 3,numerous septa without cirrhosis; and score: 4, cirrhosis.

Knodell's scoring system, also called the Hepatitis Activity Index,classifies specimens based on scores in four categories of histologicfeatures: I. Periportal and/or bridging necrosis; II. Intralobulardegeneration and focal necrosis; III. Portal inflammation; and IV.Fibrosis. In the Knodell staging system, scores are as follows: score:0, no fibrosis;

score: 1, mild fibrosis (fibrous portal expansion); score: 2, moderatefibrosis; score: 3, severe fibrosis (bridging fibrosis); and score: 4,cirrhosis. The higher the score, the more severe the liver tissuedamage. Knodell (1981) Hepatol. 1:431.

In the Scheuer scoring system scores are as follows: score: 0, nofibrosis; score: 1, enlarged, fibrotic portal tracts; score: 2,periportal or portal-portal septa, but intact architecture; score: 3,fibrosis with architectural distortion, but no obvious cirrhosis; score:4, probable or definite cirrhosis. Scheuer (1991) J. Hepatol. 13:372.

The Ishak scoring system is described in Ishak (1995) J. Hepatol.22:696-699. Stage 0, No fibrosis; Stage 1, Fibrous expansion of someportal areas, with or without short fibrous septa; stage 2, Fibrousexpansion of most portal areas, with or without short fibrous septa;stage 3, Fibrous expansion of most portal areas with occasional portalto portal (P—P) bridging; stage 4, Fibrous expansion of portal areaswith marked bridging (P—P) as well as portal-central (P—C); stage 5,Marked bridging (P—P and/or P—C) with occasional nodules (incompletecirrhosis); stage 6, Cirrhosis, probable or definite. The benefit ofanti-fibrotic therapy can also be measured and assessed by using theChild-Pugh scoring system which comprises a multicomponent point systembased upon abnormalities in serum bilirubin level, serum albumin level,prothrombin time, the presence and severity of ascites, and the presenceand severity of encephalopathy. Based upon the presence and severity ofabnormality of these parameters, patients may be placed in one of threecategories of increasing severity of clinical disease: A, B, or C.

In some embodiments, a therapeutically effective combination of IFN-γand pirfenidone or a specific pirfenidone analog is a combined dosageamount of IFN-γ and pirfenidone or a specific pirfenidone analog thateffects a change of one unit or more in the fibrosis stage based on pre-and post-therapy liver biopsies. In particular embodiments, atherapeutically effective combination of IFN-γ and pirfenidone or aspecific pirfenidone analog reduces liver fibrosis by at least one unitin the METAVIR, the Knodell, the Scheuer, the Ludwig, or the Ishakscoring system.

Secondary, or indirect, indices of liver function can also be used toevaluate the efficacy of IFN-γ and pirfenidone or specific pirfenidoneanalog combination treatment. Morphometric computerized semi-automatedassessment of the quantitative degree of liver fibrosis based uponspecific staining of collagen and/or serum markers of liver fibrosis canalso be measured as an indication of the efficacy of a subject treatmentmethod. Secondary indices of liver function include, but are not limitedto, serum transaminase levels, prothrombin time, bilirubin, plateletcount, portal pressure, albumin level, and assessment of the Child-Pughscore.

In another embodiment, an effective combination of IFN-γ and pirfenidoneor a specific pirfenidone analog is an amount of IFN-γ and an amount ofpirfenidone or a specific pirfenidone analog that in combination areeffective to increase an index of liver function by at least about 10%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, or at least about 80%, or more, comparedto the index of liver function in an untreated individual, or in aplacebo-treated individual. Those skilled in the art can readily measuresuch indices of liver function, using standard assay methods, many ofwhich are commercially available, and are used routinely in clinicalsettings.

Serum markers of liver fibrosis can also be measured as an indication ofthe efficacy of a subject treatment method. Serum markers of liverfibrosis include, but are not limited to, hyaluronate, N-terminalprocollagen III peptide, 7S domain of type IV collagen, C-terminalprocoilagen I peptide, and laminin. Additional biochemical markers ofliver fibrosis include α-2-macroglobulin, haptoglobin, gamma globulin,apolipoprotein A, and gamma glutamyl transpeptidase.

In another embodiment, a therapeutically effective combination of IFN-γand pirfenidone or a specific pirfenidone analog is an amount of IFN-γand an amount of pirfenidone or a specific pirfenidone analog that incombination are effective to reduce a serum level of a marker of liverfibrosis by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, or at leastabout 80%, or more, compared to the level of the marker in an untreatedindividual, or in a placebo-treated individual. Those skilled in the artcan readily measure such serum markers of liver fibrosis, using standardassay methods, many of which are commercially available, and are usedroutinely in clinical settings. Methods of measuring serum markersinclude immunological-based methods, e.g., eruyme-linked immunosorbentassays (ELISA), radioimmunoassays, and the like, using antibody specificfor a given serum marker.

Quantitative tests of functional liver reserve can also be used toassess the efficacy of treatment with IFN-γ. These include: indocyaninegreen clearance (ICG), galactose elimination capacity (GEC), aminopyrinebreath test (ABT), antipyrine clearance, monoethylglycine-xylidide(MEG-X) clearance, and caffeine clearance.

As used herein, a “complication associated with cirrhosis of the liver”refers to a disorder that is a sequellae of decompensated liver disease,i.e., or occurs subsequently to and as a result of development of liverfibrosis, and includes, but it not limited to, development of ascites,variceal bleeding, portal hypertension, jaundice, progressive liverinsufficiency, encephalopathy, hepatocellular carcinoma, liver failurerequiring liver transplantation, and liver-related mortality.

In another embodiment, a therapeutically effective combination of IFN-γand pirfenidone or a specific pirfenidone analog is an amount of IFN-γand an amount of pirfenidone or specific pirfenidone analog that incombination are effective in reducing the incidence (e.g., thelikelihood that an individual will develop) of a disorder associatedwith cirrhosis of the liver by at least about 10%, at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, or at least about 80%, or more, compared to an untreatedindividual, or in a placebo-treated individual.

Whether combination therapy with IFN-γ and pirfenidone or a specificpirfenidone analog is effective in reducing the incidence of a disorderassociated with cirrhosis of the liver can readily be determined bythose skilled in the art.

Reduction in liver fibrosis increases liver function. Thus, theinvention provides methods for increasing liver function, generallyinvolving administering a therapeutically effective combination of IFN-γand pirfenidone or a specific pirfenidone analog. Liver fumctionsinclude, but are not limited to, synthesis of proteins such as serumproteins (e.g., albumin, clotting factors, alkaline phosphatase,aminotrnsferases (e.g., alanine transaminase, aspartate transaminase),5′-nucleosidase, γ-glutauinyltranspeptidase, etc.), synthesis ofbilirubin, synthesis of cholesterol, and synthesis of bile acids; aliver metabolic function, including, but not limited to, carbohydratemetabolism, amino acid and ammonia metabolism, hormone metabolism, andlipid metabolism; detoxification of exogenous drugs; a hemodynamicfunction, including splanchnic and portal hemodynamics; and the like.

Whether a liver function is increased is readily ascertainable by thoseskilled in the art, using well-established tests of liver function.Thus, synthesis of markers of liver function such as albumin, alkalinephosphatase, alanine transaminase, aspartate transaminase, bilirubin,and the like, can be assessed by measuring the level of these markers inthe serum, using standard immunological and enzymatic assays. Splanchniccirculation and portal hemodynamics can be measured by portal wedgepressure and/or resistance using standard methods. Metabolic functionscan be measured by measuring the level of ammonia in the serum.

Whether serum proteins normally secreted by the liver are in the normalrange can be determined by measuring the levels of such proteins, usingstandard immunological and enzymatic assays. Those skilled in the artknow the normal ranges for such serum proteins. The following arenon-limiting examples. The normal range of alanine transaminase is fromabout 7 to about 56 units per liter of serum. The normal range ofaspartate transaminase is from about 5 to about 40 units per liter ofserum. Bilirubin is measured using standard assays. Normal bilirubinlevels are usually less than about 1.2 mg/dL. Serum albumin levels aremeasured using standard assays. Normal levels of serum albumin are inthe range of from about 35 to about 55 g/L. Prolongation of prothrombintime is measured using standard assays. Normal prothrombin time is lessthan about 4 seconds longer than control.

In another embodiment, a therapeutically effective combination of IFN-γwith pirfenidone or a specific pirfenidone analog is one that iseffective to increase liver function by at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, or more. Forexample, a therapeutically effective combination of IFNγ and pirfenidoneor a specific pirfenidone analog is a combined dosage effective toreduce an elevated level of a serum marker of liver function by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, or more, or to reduce the level of the serum marker of liverfunction to within a normal range. A therapeutically effectivecombination of IFNγ with pirfenidone or a specific pirfenidone analog isalso an amount effective to increase a reduced level of a serum markerof liver function by at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, or more, or to increase thelevel of the serum marker of liver flnction to within a normal range.

Methods of Treating Renal Fibrosis

Renal fibrosis is characterized by the excessive accumulation ofextracellular matrix (ECM) components. Overproduction of transforminggrowth factor-beta (TGF-β) is believed to underly tissue fibrosis causedby excess deposition of ECM, resulting in disease. TGF-β's fibrogenicaction results from simultaneous stimulation of matrix proteinsynthesis, inhibition of matrix degradation and enhanced integrinexpression that facilitates ECM assembly.

The present invention provides methods of treating renal fibrosis. Themethods generally involve administering to an individual having renalfibrosis a combination of an effective amount of IFN-γ and an effectiveamount of pirfenidone or a specific pirfenidone analog. As used herein,an “effective amount” of IFN-γ in combination with an “effective amount”of pirfenidone or specific pirfenidone analog is a combined dosage ofIFN-γ and pirfenidone or specific pirfenidone analog that is effectivein reducing renal fibrosis; and/or that is effective in reducing thelikelihood that an individual will develop renal fibrosis; and/or thatis effective in reducing a parameter associated with renal fibrosis;and/or that is effective in reducing a disorder associated with fibrosisof the kidney.

In one embodiment, an effective combination of IFN-γ and pirfenidone ora specific pirfenidone analog is an amount of IFN-γ and an amount ofpirfenidone or a pirfenidone analog that in combination are sufficientto reduce renal fibrosis by at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%,compared to the degree of renal fibrosis in the individual prior totreatment with the combination therapy of the present invention.

Whether fibrosis is reduced in the kidney is determined using any knownmethod. For example, histochemical analysis of kidney biopsy samples forthe extent of ECM deposition and/or fibrosis is performed. Other methodsare known in the art. See, e.g., Masseroli et al. (1998) Lab. Invest.78:511-522; U.S. Pat. No. 6,214,542.

In some embodiments, an effective combination of IFN-γ and pirfenidoneor a specific pirfenidone analog is an amount of IFN-γ and an amount ofpirfenidone or a specific pirfenidone analog that in combination areeffective to increase kidney function by at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, compared to the basal level of kidney function in theindividual prior to treatment with the combination therapy of thepresent invention.

In some embodiments, an effective combination of IFN-γ and pirfenidoneor a specific pirfenidone analog is an amount of IFN-γ and an amount ofpirfenidone or a specific pirfenidone analog that in combination areeffective to slow the decline in kidney function by at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, compared to the decline in kidney function thatwould occur in the absence of treatment with the combination therapy ofthe present invention.

Kidney function can be measured using any known assay, including, butnot limited to, plasma creatinine level (where normal levels aregenerally in a range of from about 0.6 to about 1.2 mg/dL); creatinineclearance (where the normal range for creatinine clearance is from about97 to about 137 mL/minute in men, and from about 88 to about 128mL/minute in women); the glomerular filtration rate (either calculatedor obtained from inulin clearance or other methods), blood urea nitrogen(where the normal range is from about 7 to about 20 mg/dL); and urineprotein levels.

In other embodiments, the present invention provides methods thatinvolve administering a synergistic combination of IFN-γ and pirfenidoneor a specific pirfenidone analog. As used herein, a “synergisticcombination” of IFN-γ and pirfenidone or a specific pirfenidone analogis a combined dosage that is more effective in the therapeutic orprophylactic treatment of renal fibrosis than the incrementalimprovement in treatment outcome that could be predicted or expectedfrom a merely additive combination of (i) the therapeutic orprophylactic benefit of IFN-γ when administered at that same dosage as amonotherapy and (ii) the therapeutic or prophylactic benefit ofpirfenidone or a specific pirfenidone analog when administered at thesame dosage as a monotherapy.

The invention also provides a method for treatment of renal fibrosis inan individual comprising administering to the individual a combinationof IFN-γ and pirfenidone or a specific pirfenidone analog that iseffective for prophylaxis or therapy of renal fibrosis in theindividual, e.g., increasing the time to doubling of serum creatininelevels, increasing the time to end-stage renal disease requiring renalreplacement therapy (e.g., dialysis or transplant), increasing theprobability of survival, reducing the risk of death, ameliorating thedisease burden or slowing the progression of disease in the individual,while reducing the incidence or severity of one or more side effectsthat would ordinarily arise from treatment with an effective amount ofIFN-γ or pirfenidone or a specific pirfenidone analog alone.

Without being limited by the following description, the mode ofoperation of the active ingredients of the combination therapy of theinvention is postulated to be the following. The IFN-γ may modify immuneaction, regulating the synthesis of other cytokines such as TGFβ andinhibiting fibroblast proliferation and or migration. Pirfenidone, onthe other hand, may effectively inhibit the synthesis and deposition ofECM by activated fibroblasts. In either case, the end point will be thesame, namely the therapeutic or prophylactic treatment of fibroticdisease.

Pirfenidone and Analogs Thereof

Pirfenidone(5-methyl-1-phenyl-2-(1H)-pyridone) and specific pirfenidoneanalogs are disclosed for the treatment of fibrotic conditions. A“fibrotic condition” is one that is amenable to treatment byadministration of a compound having anti-fibrotic activity.

Descriptions for Substituents R₁, R₂, X

R₁: carbocyclic (saturated and unsaturated), heterocyclic (saturated orunsaturated), alkyls (saturated and unsaturated). Examples includephenyl, benzyl, pyrimidyl, naphthyl, indolyl, pyrrolyl, furyl, thienyl,imidazolyl, cyclohexyl, piperidyl, pyrrolidyl, morpholinyl,cyclohexenyl, butadienyl, and the like.

R₁ can further include substitutions on the carbocyclic or heterocyclicmoieties with substituents such as halogen, nitro, amino, hydroxyl,alkoxy, carboxyl, cyano, thio, alkyl, aryl, heteroalkyl, heteroaryl andcombinations thereof, for example, 4-nitrophenyl, 3-chlorophenyl,2,5-dinitrophenyl, 4-methoxyphenyl, 5-methyl-pyrrolyl,2,5-dichlorocyclohexyl, guanidinyl-cyclohexenyl and the like.

R₂: alkyl, carbocylic, aryl, heterocyclic. Examples include: methyl,ethyl, propyl, isopropyl, phenyl, 4-nitrophenyl, thienyl and the like.

X: may be any number (from 1 to 3) of substituents on the carbocyclic orheterocyclic ring. The substituents can be the same or different.Substituents can include hydrogen, alkyl, heteroalkyl, aryl, heteroaryl,halo, nitro, carboxyl, hydroxyl, cyano, amino, thio, alkylamino,haloaryl and the like.

The substituents may be optionally further substituted with 1-3substituents from the group consisting of alkyl, aryl, nitro, alkoxy,hydroxyl and halo groups. Examples include: methyl, 2,3-dimethyl,phenyl, p-tolyl, 4-chlorophenyl, 4-nitrophenyl, 2,5-dichlorophenyl,furyl, thienyl and the like.

Specific Examples include: TABLE 1 IA IIB 5-Methyl-1-(2′-pyridyl)-2-(1H)pyridine, 6-Methyl-1-phenyl-3-(1H) pyridone, 6-Methyl-1-phenyl-2-(1H)pyridone, 5-Methyl-1-p-tolyl-3-(1H) pyridone,5-Methyl-3-phenyl-1-(2′-thienyl)-2-(1H) 5-Methyl-1-(2′-naphthyl)-3-(1H)pyridone, pyridone, 5-Methyl-1-(2′-naphthyl)-2-(1H) pyridone,5-Methyl-1-phenyl-3-(1H) pyridone, 5-Methyl-1-p-tolyl-2-(1H) pyridone,5-Methyl-1-(5′-quinolyl)-3-(1H) pyridone, 5-Methyl-1-(1′naphthyl)-2-(1H)pyridone, 5-Ethyl-1-phenyl-3-(1H) pyridone, 5-Ethyl-1-phenyl-2-(1H)pyridone, 5-Methyl-1-(4′-methoxyphenyl)-3-(1H) pyridone,5-Methyl-1-(5′-quinolyl)-2-(1H) pyridone, 4-Methyl-1-phenyl-3-(1H)pyridone, 5-Methyl-1-(4′-quinolyl)-2-(1H) pyridone,5-Methyl-1-(3′-pyridyl)-3-(1H) pyridone, 5-Methyl-1-(4′-pyridyl)-2-(1H)pyridone, 5-Methyl-1-(2′-Thienyl)-3-(1H) pyridone,3-Methyl-1-phenyl-2-(1H) pyridone, 5-Methyl-1-(2′-pyridyl)-3-(1H)pyridone, 5-Methyl-1-(4′-methoxyphenyl)-2-(1H)5-Methyl-1-(2′-quinolyl)-3-(1H) pyridone, pyridone, 1-Phenyl-2-(1H)pyridone, 1-Phenyl-3-(1H) pyridine, 1,3-Diphenyl-2-(1H) pyridone,1-(2′-Furyl)-5-methyl-3-(1H) pyridone, 1,3-Diphenyl-5-methyl-2-(1H)pyridone, 1-(4′-Chlorophenyl)-5-methyl-3-(1H) pyridine.5-Methyl-1-(3′-trifluoromethylphenyl)-2- (1H)-pyridone,3-Ethyl-1-phenyl-2-(1H) pyridone, 5-Methyl-1-(3′-pyridyl)-2-(1H)pyridone, 5-Methyl-1-(3-nitrophenyl)-2-(1H) pyridone,3-(4′-Chlorophenyl)-5-Methyl-1-phenyl-2- (1H) pyridone,5-Methyl-1-(2′-Thienyl)-2-(1H) pyridone,5-Methyl-1-(2′-thiazolyl)-2-(1H) pyridone, 3,6-Dimethyl-1-phenyl-2-(1H)pyridone, 1-(4′Chlorophenyl)-5-Methyl-2-(1H) pyridone,1-(2′-Imidazolyl)-5-Methyl-2-(1H) pyridone, 1-(4′-Nitrophenyl)-2-(1H)pyridone, 1-(2′-Furyl)-5-Methyl-2-(1H) pyridone,1-Phenyl-3-(4′-chlorophenyl)-2-(1H) pyridine.

U.S. Pat. Nos. 3,974,281; 3,839,346; 4,042,699; 4,052,509; 5,310,562;5,518,729; 5,716,632; and 6,090,822 describe methods for the synthesisand formulation of pirfenidone and specific pirfenidone analogs inpharmaceutical compositions suitable for use in the methods of thepresent invention.

Interferon-Gamma

The nucleic acid sequences encoding IFN-γ polypeptides may be accessedfrom public databases, e.g. Genbank, journal publications, etc. Whilevarious mammalian IFN-γ polypeptides are of interest, for the treatmentof human disease, generally the human protein will be used. Human IFN-γcoding sequence may be found in Genbank, accession numbers X13274;V00543; and NM_(—)000619. The corresponding genomic sequence may befound in Genbank, accession numbers J00219; M37265; and V00536. See, forexample. Gray et al. (1982) Nature 295:501 (Genbank X13274); andRinderknecht et al. (1984) J. Biol. Chem. 259:6790.

IFN-γ1b (Actimmune®; human interferon) is a single-chain polypeptide of140 amino acids. It is made recombinantly in E. coli and isunglycosylated. Rinderknecht et al. (1984) J. Biol. Chem. 259:6790-6797.

The IFN-γ to be used in the compositions of the present invention may beany of natural IFN-γs, recombinant IFN-γs and the derivatives thereof sofar as they have a IFN-γ activity, particularly human IFN-γ activity.Human IFN-γ exhibits the antiviral and anti-proliferative propertiescharacteristic of the interferons, as well as a number of otherimmunomodulatory activities, as is known in the art. Although IFN-γ isbased on the sequences as provided above, the production of the proteinand proteolytic processing can result in processing variants thereof.The unprocessed sequence provided by Gray et al., supra. consists of 166amino acids (aa). Although the recombinant IFN-γ produced in E. coli wasoriginally believed to be 146 amino acids, (commencing at amino acid 20)it was subsequently found that native human IFN-γ is cleaved afterresidue 23, to produce a 143 aa protein, or 144 aa if the terminalmethionine is present, as required for expression in bacteria Duringpurification, the mature protein can additionally be cleaved at the Cterminus after reside 162 (referring to the Gray et al. sequence),resulting in a protein of 139 amino acids, or 140 amino acids if theinitial methionine is present, e.g. if required for bacterialexpression. The N-terminal methionine is an artifact encoded by the mRNAtranslational “start” signal AUG which, in the particular case of E.coli expression is not processed away. In other microbial systems oreukaryotic expression systems, methionine may be removed.

For use in the subject methods, any of the native IFN-γ peptides,modifications and variants thereof, or a combination of one or morepeptides may be used. IFN-γ peptides of interest include fragments, andcan be variously truncated at the carboxy terminal end relative to thefull sequence. Such fragments continue to exhibit the characteristicproperties of human gamma interferon, so long as amino acids 24 to about149 (numbering from the residues of the unprocessed polypeptide) arepresent. Extraneous sequences can be substituted for the amino acidsequence following amino acid 155 without loss of activity. See, forexample, U.S. Pat. No. 5,690,925, herein incorporated by reference.Native IFN-γ moieties include molecules variously extending from aminoacid residues 24-150; 24-151, 24-152; 24- 153, 24-155; and 24-157. Anyof these variants, and other variants known in the art and having IFN-γactivity, may be used in the present methods.

The sequence of the IFN-γ polypeptide may be altered in various waysknown in the art to generate targeted changes in sequence. A variantpolypeptide will usually be substantially similar to the sequencesprovided herein, i.e. will differ by at least one amino acid, and maydiffer by at least two but not more than about ten amino acids. Thesequence changes may be substitutions, insertions or deletions. Scanningmutations that systematically introduce alanine, or other residues, maybe used to determine key amino acids. Specific amino acid substitutionsof interest include conservative and non-conservative changes.Conservative amino acid substitutions typically include substitutionswithin the following groups: (glycine, alanine); (valine, isoleucine,leucine); (aspartic acid, glutamic acid); (asparagine, glutamine);(serine, threonine); (lysine, arginine); or (phenylalanine, tyrosine).

Modifications of interest that may or may not alter the primary aminoacid sequence include chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation; changes in amino acid sequence thatintroduce or remove a glycosylation site; changes in amino acid sequencethat make the protein susceptible to PEGylation; and the like. In oneembodiment, the invention contemplates the use of IFN-γ variants withone or more non-naturally occurring glycosylation and/or pegylationsites that are engineered to provide glycosyl- and/or PEG-derivatizedpolypeptides with reduced serum clearance, such as the IFN-γ polypeptidevariants described in International Patent Publication No. WO 01/36001.Also included are modifications of glycosylation, e.g. those made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing or in further processing steps; e.g. byexposing the polypeptide to enzymes that affect glycosylation, such asmammalian glycosylating or deglycosylating enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.phosphotyrosine, phosphoserine, or phosphothreonine.

Included in the subject invention are polypeptides that have beenmodified using ordinary chemical techniques so as to improve theirresistance to proteolytic degradation, to optimize solubilityproperties, or to render them more suitable as a therapeutic agent. Forexamples, the backbone of the peptide may be cyclized to enhancestability (see Friedler et al. (2000) J. Biol. Chem. 275:23783-23789).Analogs may be used that include residues other than naturally occurringL-amino acids, e.g. D-amino acids or non-naturally occurring syntheticamino acids. The protein may be pegylated to enhance stability.

The polypeptides may be prepared by in vitro synthesis, usingconventional methods as known in the art, by recombinant methods, or maybe isolated from cells induced or naturally producing the protein. Theparticular sequence and the manner of preparation will be determined byconvenience, economics, purity required, and the like. If desired,various groups may be introduced into the polypeptide during synthesisor during expression, which allow for linking to other molecules or to asurface. Thus cysteines can be used to make thioethers, histidines forlinking to a metal ion complex, carboxyl groups for forming amides oresters, amino groups for forming amides, and the like.

The polypeptides may also be isolated and purified in accordance withconventional methods of recombinant synthesis. A lysate may be preparedof the expression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. For the most part, the compositions which areused will comprise at least 20% by weight of the desired product, moreusually at least about 75% by weight, preferably at least about 95% byweight, and for therapeutic purposes, usually at least about 99.5% byweight, in relation to contaminants related to the method of preparationof the product and its purification. Usually, the percentages will bebased upon total protein.

Dosages, Formulations, and Routes of Administration

IFN-γ and pirfenidone or specific pirfenidone analogs are administeredto individuals in a formulation (e.g., in separate formulations) with apharmaceutically acceptable excipient(s). A wide variety ofpharmaceutically acceptable excipients are known in the art and need notbe discussed in detail herein. Pharmaceutically acceptable excipientshave been amply described in a variety of publications, including, forexample, A. Gennaro (2000) “Remington: The Science and Practice ofPharmacy”, 20th edition, Lippincott, Williams, & Wilkins; PharmaceuticalDosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds7^(th) ed., Lippincott, Williams, & Wilkins; and Handbook ofPharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed.Amer. Pharmaceutical Assoc.

In the subject methods, the active agent(s) may be administered to thehost using any convenient means capable of resulting in the desiredtherapeutic effect. Thus, the agent can be incorporated into a varietyof formulations for therapeutic administration. More particularly, theagents of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants and aerosols.

As such, administration of the agents can be achieved in various ways,including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, etc., administration.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Effective dosages of IFN-γ can range from about 0.5 μg/m² to about 500μg/m², usually from about 1.5 μg/m² to 200 μg/m², depending on the sizeof the patient. This activity is based on 10⁶ international units (IU)per 50 μg of protein.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven compound are readily determinable by those of skill in the art bya variety of means. A preferred means is to measure the physiologicalpotency of a given compound.

In specific embodiments of interest, IFN-γ is administered to anindividual in a unit dosage form of from about 25 μg to about 500 μg,from about 50 μg to about 400 μg, or from about 100 μg to about 300 μg.In particular embodiments of interest, the dose is about 200 μg IFN-γ.In many embodiments of interest, IFN-γ1b is administered.

Where the dosage is 200 μg IFN-γ per dose, the amount of IFN-γ per bodyweight (assuming a range of body weights of from about 45 kg to about135 kg) is in the range of from about 4.4 μg IFN-γ per kg body weight toabout 1.48 μg IFN-γ per kg body weight.

The body surface area of subject individuals generally ranges from about1.33 m² to about 2.50 m². Thus, in many embodiments, an IFN-γ dosageranges from about 150 μg/m² to about 20 μg/m². For example, an IFN-γdosage ranges from about 20 μg/m² to about 30 μg/m², from about 30 μg/m²to about 40 μg/m², from about 40 μg/m² to about 50 μg/m², from about 50μg/m² to about 60 μg/m², from about 60 μg/m² to about 70 μg/m², fromabout 70 μg/m² to about 80 μg/m², from about 80 μg/m² to about 90 μg/m²,from about 90 μg/m² to about 100 μg/m², from about 100 μg/m² to about110 μg/m², from about 110 μg/m² to about 120 μg/m², from about 120 μg/m²to about 130 μg/m², from about 130 μg/m² to about 140 μg/m², or fromabout 140 μg/m² to about 150 μg/m². In some embodiments, the dosagegroups range from about 25 μg/m² to about 100 μg/m². In otherembodiments, the dosage groups range from about 25 μg/m² to about 50μg/m²,

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Where the agent is a polypeptide, polynucleotide (e.g., a polynucleotideencoding IFN-γ), it may be introduced into tissues or host cells by anynumber of routes, including viral infection, microinjection, or fusionof vesicles. Jet injection may also be used for intramuscularadministration, as described by Furth et al. (1992), Anal Biochem205:365-368. The DNA may be coated onto gold microparticles, anddelivered intradermally by a particle bombardment device, or “gene gun”as described in the literature (see, for example, Tang et al. (1992),Nature 356:152-154), where gold microprojectiles are coated with thetherapeutic DNA, then bombarded into skin cells. Of particular interestin these embodiments is use of a liver-specific promoter to drivetranscription of an operably linked IFN-γ coding sequence preferentiallyin liver cells.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

In particular embodiments of interest, IFN-γ is administered as asolution suitable for subcutaneous injection. For example, IFN-γ is in aformulation containing 40 mg mannitol/mL, 0.72 mg sodium succinate/mL,0.10 mg polysorbate 20/mL. In particular embodiments of interest, IFN-γis administered in single-dose forms of 200 μg/dose subcutaneously.

Multiple doses of IFN-γ can be administered, e.g., IFN-γ can beadministered once per month, twice per month, three times per month,once per week, twice per week, three times per week, four times perweek, five times per week, six times per week, or daily, over a periodof time ranging from about one day to about one week, from about twoweeks to about four weeks, from about one month to about two months,from about two months to about four months, from about four months toabout six months, from about six months to about eight months, fromabout eight months to about 1 year, from about 1 year to about 2 years,or from about 2 years to about 4 years, or more. In particularembodiments of interest, IFN-γ is administered three times per week overa period of about 48 weeks.

Pirfenidone can be administered once per month, twice per month, threetimes per month, once per week, twice per week, three times per week,four times per week, five times per week, six times per week, daily, orin divided daily doses ranging from once daily to 5 times daily over aperiod of time ranging from about one day to about one week, from abouttwo weeks to about four weeks, from about one month to about two months,from about two months to about four months, from about four months toabout six months, from about six months to about eight months, fromabout eight months to about 1 year, from about 1 year to about 2 years,or from about 2 years to about 4 years, or more.

Effective dosages of pirfenidone or specific pirfenidone analogs willrange from about 5 mg/kg/day to about 125 mg/kg/day, or at a fixeddosage of about 400 mg to about 3600 mg per day, administered orally.Other doses and formulations of pirfenidone and specific pirfenidoneanalogs suitable for use in the treatment of fibrotic diseases aredescribed in U.S. Pat. Nos. 3,974,281; 3,839,346; 4,042,699; 4,052,509;5,310,562; 5,518,729; 5,716,632; and 6,090,822.

IFN-γ and pirfenidone (or pirfenidone analog) are generally administeredin separate formulations. IFN-γ and pirfenidone (or pirfenidone analog)may be administered substantially simultaneously, or within about 30minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours,about 16 hours, about 24 hours, about 36 hours, about 72 hours, about 4days, about 7 days, or about 2 weeks of one another.

In one embodiment, the invention provides a method for treatment of afibrotic disease in an individual comprising administering to theindividual a combination therapy comprising about 25 mcg/m² (25 μg/m²)to about 50 mcg/m² IFN-γ three times weekly and about 2400 mg to about3600 mg pirfenidone or a specific pirfenidone analog daily. In anotherembodiment, the invention provides a method for treatment of a fibroticdisease in an individual comprising administering to the individual acombined dosage of about 50 μg to about 100 μg IFN-γ three times weeklyand about 400 mg to about 2400 mg pirfenidone or a specific pirfenidoneanalog daily.

In one embodiment, the invention provides a method for treatment of IPFin an individual comprising administering to the individual acombination therapy comprising about 25 mcg/m² to about 50 mcg/m² IFN-γthree times weekly and about 2400 mg to about 3600 mg pirfenidone or aspecific pirfenidone analog daily. In another embodiment, the inventionprovides a method for treatment of IPF in an individual comprisingadministering to the individual a combined dosage of about 50 μg toabout 100 μg IFN-γ three times weekly and about 400 mg to about 2400 mgpirfenidone or a specific pirfenidone analog daily.

In one embodiment, the invention provides a method for treatment ofliver fibrosis in an individual comprising administering to theindividual a combination therapy comprising about 25 mcg/m² to about 50mcg/m² IFN-γ three times weekly and about 2400 mg to about 3600 mgpirfenidone or a specific pirfenidone analog daily. In anotherembodiment, the invention provides a method for treatment of liverfibrosis in an individual comprising administering to the individual acombined dosage of about 50 μg to about 100 μg IFN-γ three times weeklyand about 400 mg to about 2400 mg pirfenidone or a specific pirfenidoneanalog daily.

In one embodiment, the invention provides a method for treatment ofrenal fibrosis in an individual comprising administering to theindividual a combination therapy comprising about 25 mcg/r² to about 50mcg/m² IFN-γ three times weekly and about 2400 mg to about 3600 mgpirfenidone or a specific pirfenidone analog daily. In anotherembodiment, the invention provides a method for treatment of renalfibrosis in an individual comprising administering to the individual acombined dosage of about 50 μg to about 100 μg IFN-γ three times weeklyand about 400 mg to about 2400 mg pirfenidone or a specific pirfenidoneanalog daily.

In an exemplary embodiment, the invention provides a method fortreatment of a fibrotic disease in an individual, the method comprisingadministering to an individual having a fibrotic disease IFN-γ in anamount of 200 μg three times per week subcutaneously; and pirfenidone ora pirfenidone analog in an amount of 1800 mg/day to 3600 mg/day, in asingle dose or in two or three divided doses administered orally perday, for a period of time of 2-5 years. In another exemplary embodiment,the invention provides a method for treatment of a fibrotic disease inan individual, the method comprising administering to an individualhaving a fibrotic disease IFN-γ in an amount of 200 μg three times perweek subcutaneously; and pirfenidone or a pirfenidone analog in anamount of 1200 mg daily, in a single dose or in two or three divideddoses administered orally per day, for a period of time of 2-5 years.For example, IFN-γ is administered as a solution suitable forsubcutaneous injection. For example, IFN-γ is in a formulationcontaining 40 mg mannitol/mL, 0.72 mg sodium succinate/mL, 0.10 mgpolysorbate 20/mL.

In another embodiment, the invention provides a method for treatment offibrotic disease in an individual comprising administering to theindividual a synergistic combination of (i) about IFN-γ 25 mcg/m² toabout 100 mcg/m² IFN-γ, or about 50 μg IFN-γ to about 200 μg IFN-γ,administered subcutaneously three times per week and (ii) about 400 mgto about 3600 mg pirfenidone or a specific pirfenidone analog in asingle dose or two or three divided doses administered orally per day.

In another exemplary embodiment, the invention provides a method fortreatment of a IPF in an individual, the method comprising administeringto an individual having IPF IFN-γ in an amount of 200 μg three times perweek subcutaneously; and pirfenidone or a pirfenidone analog in anamount of 1800 mg/day to 3600 mg/day, in a single dose or in two orthree divided doses administered orally per day, for a period of time of2-5 years. In yet another exemplary embodiment, the invention provides amethod for treatment of IPF in an individual, the method comprisingadministering to an individual having IPF IFN-γ in an amount of 200 μgthree times per week subcutaneously; and pirfenidone or a pirfenidoneanalog in an amount of 1200 mg daily, in a single dose or in two orthree divided doses administered orally per day, for a period of time of2-5 years.

In another embodiment, the invention provides a method for treatment ofIPF in an individual comprising administering to the individual asynergistic combination of (i) about IFN-γ 25 μg/m² to about 100 μg/m²IFN-γ, or about 50 μg IFN-γ to about 200 μg IFN-γ, administeredsubcutaneously three times per week and (ii) about 400 mg to about 3600mg pirfenidone or a specific pirfenidone analog in a single dose or twoor three divided doses administered orally per day.

In an exemplary embodiment, the invention provides a method fortreatment of liver fibrosis in an individual, the method comprisingadministering to an individual having liver fibrosis IFN-γ in an amountof 200 μg three times per week subcutaneously; and pirfenidone or apirfenidone analog in an amount of 1800 mg/day to 3600 mg/day, in asingle dose or in two or three divided doses administered orally perday, for a period of time of 2-5 years. In another exemplary embodiment,the invention provides a method for treatment of liver fibrosis in anindividual, the method comprising administering to an individual havingliver fibrosis IFN-γ in an amount of 200 μg three times per weeksubcutaneously; and pirfenidone or a pirfenidone analog in an amount of1200 mg daily, in a single dose or in two or three divided dosesadministered orally per day, for a period of time of 2-5 years.

In another embodiment, the invention provides a method for treatment ofliver fibrosis in an individual comprising administering to theindividual a synergistic combination of (i) about IFN-γ 25 mcg/m² toabout 100 mcg/m² IFN-γ, or about 50 μg IFN-γ to about 200 μg IFN-γ,administered subcutaneously three times per week and (ii) about 400 mgto about 3600 mg pirfenidone or a specific pirfenidone analog in asingle dose or two or three divided doses administered orally per day.

In an exemplary embodiment, the invention provides a method fortreatment of renal fibrosis in an individual, the method comprisingadministering to an individual having renal fibrosis IFN-γ in an amountof 200 μg three times per week subcutaneously; and pirfenidone or apirfenidone analog in an amount of 1200 mg/day to 3600 mg/day, in asingle dose or in two or three divided doses administered orally perday, for a period of time of 2-5 years. In another exemplary embodiment,the invention provides a method for treatment of renal fibrosis in anindividual, the method comprising administering to an individual havingrenal fibrosis IFN-γ in an amount of 200 μg three times per weeksubcutaneously; and pirfenidone or a pirfenidone analog in an amount of800 mg daily, in a single dose or in two or three divided dosesadministered orally per day, for a period of time of 2-5 years.

In another embodiment, the invention provides a method for treatment ofrenal fibrosis in an individual comprising administering to theindividual a synergistic combination of (i) about IFN-γ 25 mcg/m² toabout 100 mcg/m² IFN-γ, or about 50 μg IFN-γ to about 200 μg IFN-γ,administered subcutaneously three times per week and (ii) about 400 mgto about 3600 mg pirfenidone or a specific pirfenidone analog in asingle dose or two or three divided doses administered orally per day.

Additional Agents

In some embodiments, IFN-γ and pirfenidone or a specific pirfenidoneanalog are co-administered with one or more additional agents. Suitableadditional agents include corticosteroids, such as prednisone. Whenco-administered with IFN-γ and pirfenidone or a specific pirfenidoneanalog in the treatment of a fibrotic disease, such as IPF, liverfibrosis, or renal fibrosis, prednisone can be administered in an amountof 7.5 mg or 15 mg daily, administered orally.

Subjects Suitable for Treatment

The subject methods are suitable for treatment of individuals diagnosedas having a fibrotic disease, such as IPF, liver fibrosis or renalfibrosis. The subject methods are also suitable for treatment ofindividuals who are at risk of developing a fibrotic disease.

Individuals with liver fibrosis who are suitable for treatment accordingto the methods of the invention include individuals who have beenclinically diagnosed with liver fibrosis, as well as individuals whohave not yet developed clinical liver fibrosis but who are considered atrisk of developing liver fibrosis. Such individuals include, but are notlimited to, individuals who are infected with HCV; individuals who areinfected with HBV; individuals who are infected with Schistosomamansoni; individuals who have been exposed to chemical agents known toresult in liver fibrosis; individuals who have been diagnosed withWilson's disease; individuals diagnosed with hemochromatosis; andindividuals with alcoholic liver disease; individuals with non-alcoholicsteatohepatitis; individuals with autoimmune hepatitis; individuals withprimary sclerosing cholangitis, primary biliary cirrhosis, oralpha-1-antitrysin deficiency.

Individuals who have been clinically diagnosed as infected with HCV areof particular interest in many embodiments. Individuals who are infectedwith HCV are identified as having HCV RNA in their blood, and/or havinganti-HCV antibody in their serum. In many embodiments, individuals ofinterest include those who exhibit severe fibrosis or early cirrhosis(non-decompensated, Child's-Pugh class A or less), or more advancedcirrhosis (decompensated, Child's-Pugh class B or C) due to chronic HCVinfection and who are viremic despite prior anti-viral treatment withIFN-α-based therapies or who cannot tolerate IFN-α-based therapies, orwho have a contraindication to such therapies. In particular embodimentsof interest, HCV-positive individuals with stage 3 or 4 liver fibrosisaccording to the METAVIR scoring system are suitable for treatment withthe methods of the present invention. In other embodiments, individualssuitable for treatment with the methods of the instant invention arepatients with decompensated cirrhosis with clinical manifestations,including patients with far-advanced liver cirrhosis, including thoseawaiting liver transplantation. In still other embodiments, individualssuitable for treatment with the methods of the instant invention includepatients with milder degrees of fibrosis including those with earlyfibrosis (stages 1 and 2 in the METAVIR, Ludwig, and Scheuer scoringsystems; or stages 1, 2, or 3 in the Ishak scoring system.).

The subject methods are suitable for treatment of individuals diagnosedas having IPF. The methods are also suitable for treatment ofindividuals having IPF who were previously treated with corticosteroidswithin the previous 24 months, and who failed to respond to previoustreatment with corticosteroids. Also included are subjects who have anFVC at the outset of treatment that is at least 55% of the predictedFVC. The percent predicted FVC values are based on normal values, whichare known in the art. See, e.g., Crapo et al. (1981) Am. Rev. Respir.Dis. 123:659-664. FVC is measured using-standard methods of spirometry.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of treating a fibrotic disease in an individual, the methodcomprising administering to an individual suffering from a fibroticdisease a synergistic combination of IFN-γ and pirfenidone or a specificpirfenidone analog that is effective in the treatment or prophylaxis ofthe fibrotic disease in the individual.
 2. A method of treating afibrotic disease in an individual, the method comprising administeringto an individual suffering from a fibrotic disease a combination ofIFN-γ and pirfenidone or a specific pirfenidone analog that is effectivein the treatment or prophylaxis of the fibrotic disease in theindividual and that reduces the incidence or severity of one or moreside effects ordinarily associated with the administration of eitherdrug alone in the treatment of the fibrotic disease.
 3. A method oftreating a fibrotic disease in an individual, the method comprisingadministering to an individual suffering from a fibrotic disease acombined dosage of about 50 mcg to about 200 mcg IFN-γ three timesweekly and about 400 mg to about 3600 mg pirfenidone or a specificpirfenidone analog daily.
 4. The method of any of claims 1-3 wherein thefibrotic disease is pulmonary fibrosis.
 5. The method of claim 4 whereinthe pulmonary fibrosis is idiopathic pulmonary fibrosis.
 6. The methodof claim 4 wherein the pulmonary fibrosis is from a known etiology. 7.The method of any of claims 1-3 wherein the fibrotic disease is liverfibrosis.
 8. The method of any of claims 1-3 wherein the fibroticdisease is renal fibrosis.
 9. The method of any of claims 1-3 whereinthe fibrotic disease is cardiac fibrosis.
 10. The method of any ofclaims 1-3 wherein the fibrotic disease is scleroderma.
 11. The methodof any of claims 1-10 wherein the individual is a human.